Monoclonal antibody against ovarian carcinoma

ABSTRACT

The present invention provides a monoclonal antibody designated SM047 against cancer, specifically ovarian carcinoma. SM047 is strongly expressed in most ovarian serous adenocarcinomas and in other female genital tract adenocarcinomas. The monoclonal antibody according to the invention is useful to detect a cancer, specifically ovarian carcinoma and/or or determine the origin of cancer. The present invention also provides a hybridoma cell producing the monoclonal antibody and an antigen which binds to the monoclonal antibody according to the present invention.

FIELD OF THE INVENTION

The present invention is directed to a monoclonal antibody againstcancer, specifically ovarian carcinoma. The present invention is alsodirected to a hybridoma cell producing the monoclonal antibody and anantigen which binds to the monoclonal antibody according to the presentinvention. The present invention relates to a method of detecting acancer, specifically ovarian carcinoma or determining the origin ofcancer and a diagnostic aids.

BACKGROUND TO THE INVENTION

Ovarian carcinoma is disease that eludes early diagnosis and has a highmortality rate. In a diagnosis of ovarian carcinoma, one of the mostcommon problem is to distinguish between a primary ovarian andcolorectal adenocarcinoma. Colorectal adenocarcinomas metastatic to theovary may closely resemble primary ovarian endometrioid or mucinousadenocarcinomas ⁽¹⁾ and the morphological distinction is difficult.

Monoclonal antibodies raised against tumour-associated antigens havebeen widely used in pathological practice, especially in thedetermination of the likely primary site of an adenocarcinoma when thisis not known clinically. In the case of ovarian cancer these antibodieshave not proven sufficiently specific and their value is limited in adiagnostic sense. Antibodies such as CA125 and human alveolar macrophage(HAM) 56 have been proposed as useful immunohistochemical markers ofovarian adenocarcinoma but immunoreactivity is often present inadenocarcinomas of other sites, limiting their diagnostic value.^((2,3)) Anticytokeratin (CK) antibodies are of value in certaindiagnostic situations, especially in the distinction between a primaryovarian adenocarcinoma (usually CK7+, CK20−) and a colonicadenocarcinoma (usually CK7−, CK20+). ^((4,5)) However, these antibodiesare of no value in the distinction between an ovarian primary and anadenocarcinoma arising in the stomach, breast, pancreas or biliary tree,since these tumours share a common anti-CK immunoprofile.

In addition, the serological test using CA125 has been used formonitoring disease progression or recurrence after treatment but it isnot sensitive enough for early detection of such tumours and gives falsepositive results in other clinical conditions. The CA125 MAb cannot beused for immunocytochemical detection in routine pathological specimen,as it does not react with the denatured antigen in these tissues.

Accordingly, a need exists to provide an improved diagnostic tool andmethod for detecting an ovarian carcinoma cell.

BRIEF DESCRIPTION OF THE INVENTION

According to one aspect of the present invention a monoclonal antibody,designated SM047, which is specifically reactive against ovariancarcinoma is provided.

More particularly, the present invention provides a monoclonal antibodywhich binds to an epitope that is displayed by a multivalent antigenassociated with the glycocalyx of ovarian carcinoma cells.

According to a further aspect of the present invention a hybridoma cellproducing a monoclonal antibody SM047 is provided.

The present invention also provides an antigen which binds to themonoclonal antibody according to the present invention.

According to a still further aspect of the present invention there isprovided a method of detecting a carcinoma or determining the origin ofcarcinoma by using monoclonal antibody SM047. The method according tothe present invention includes serological and histochemical detection.

The present invention also provides a diagnostic aid for detecting acarcinoma or determining the origin of carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SM047 immunoprecipitates immunoblotted with SM047.Staphylococcus aureus was obtained with rabbit anti-mouse immunoglobulinand SM047 Mab as described in Example 1 and added to 100 μl UWOV1 lysateor 50 μl serum from a patient with ovarian malignancy or 50 μl poolednormal serum. After 1 h on ice the particles were washed, boiled withreducing SDS sample buffer and electrophoresed in a 4.5–15% gradient.The proteins in the gel were transferred electrophoretically to PVDFmembrane which was blocked and probed with SM047 and peroxidasedetection system with chemiluminescent substrate. Lanes 1 and 4 containSM047 immunoprecipitate from UWOV1 cells, lanes 2 and 5 from a patient'sserum and lanes 3 and 6 from a pool of five normal sera. Lanes 1–3 wereprobed with SM047 and lanes 4–6 with J138 control antibody.

FIG. 2 shows SM047 immunoprecipitates probed for sialic acid residues.SM047 immunoprecipitates from UWOV1 lysate, patient's serum and poolednormal serum were prepared and SDS-PAGE and electroblotting performed.The proteins on the membrane were oxidized with metapriodate at 4° C.and labelled with biotin hydrazide according to instructions with theglycoprotein detection kit. The membrane was finally treated withstreptavidin-peroxidase and chemiluminescent substrate as forimmunoblots. Lane 1, pooled normal serum; lane 2, patient's serum; lane3 UWOV1 lysate.

FIG. 3 shows a strong positive membrane staining of ovarian seriousadenocarcinoma with SM047.

FIG. 4 shows a strong positive membrane staining of endometrioid typeendometrial adenocarcinoma with SM047.

FIG. 5 shows a weak cytoplasmic staining with SM047 in this colonicadenocarcinoma.

DETAILED DESCRIPTION OF THE INVENTION

The monoclonal antibody according to the invention, designated SM047 ismurine IgM monoclonal antibody produced from hybridoma cells derivedfrom fusion of SP2 myeloma cells with splenocytes of a mouse that hadbeen immunized with a membrane preparation of tumour (ovarian serouscystadenocarcinoma) and boosted with cells from a cell line establishedfrom a similar tumour in a different patient.

The hybridoma cell line according to the invention producing themonoclonal antibody SM047 has been deposited with the EuropeanCollection of Cell Cultures (ECACC) at Health Protection Agency, Centrefor Emergency Preparedness and Response, Porton Down, Salisbury,Wiltshire, SP4 OJG, UK, under the accession no. 01082905 in accordancewith the Budapest Treaty.

The monoclonal antibody SM047 binds to an epitope that is displayed by amultivalent antigen associated with the glycocalyx of ovarianadenocarcinoma cells. The present invention also describes SM047staining in adenocarcinomas of diverse sites in order to determinewhether the antibody is specific for ovarian adenocarcinoma and of valuein the confirmation of an ovarian origin when the site of primary tumoris unknown. As SM047 staining is specific for an ovarian primary, it isuseful in determining the origin of an adenocarcinoma when this is notcertain on clinical and pathological grounds.

SM047 is strongly expressed in most ovarian serous adenocarcinomas andin other female genital tract adenocarcinomas, with the exception ofovarian mucinous tumours. SM047 is useful in confirming the ovarianorigin of an adenocarcinoma when used as part of a larger panel. This isespecially so in the distinction between a non-mucinous ovarianadenocarcinoma, which usually exhibits strong membranous staining, and acolonic adenocarcinoma which is usually negative or exhibits weakcytoplasmic staining.

SM047 is useful for detecting an antigen in the serum of patients withovarian cancer. SM047 immunoprecipitates the tumour antigen in the seraof patients with ovarian tumours SM047 as well as a “normal” form of theantigen. However, such antigens derived from normal cells aredistinguishable from those of tumor cells by its heterogeneous andhigher electrophoretic mobility and lack of sialic acid residues.Therefore, the process of detecting an ovarian carcinoma by using SM047according to invention comprises using a detecting reagent which reactsprimarily with the sialic acid-containing tumour-associated SM047antigen but not or only minimally with the normal form.

The monoclonal antibody SM047 according to the invention is alsosuitable for immunohistological detection of an ovarian carcinoma. Theepitope is not denatured by the formalin fixation and wax embeddingprocedure to which routine pathology specimens are subjected and thischaracteristic makes the antibody a useful diagnostic reagent forpathologists in the identification tumours.

The antigen which binds to the monoclonal antibody according to theinvention has an electrophorectic mobility of >200 kDa, andelectrophoretically homogeneous when associated with tumours, butheterogeneous and of higher mobility in sera of normal individuals. Andthe antigen has sialic acid residues on the tumour-associated moleculesand a fraction of those present in the sera from patients with tumoursbut not on the molecules present in sera from normal individuals, andhas sulphation of the tumour-associated form.

EXAMPLE 1 Development and Characterization of SM047

a. Producing and Purifying SM047

The UWOV1 cell line⁽⁶⁾ was kindly supplied by Dr T. Golombick of theUniversity of the Witwatersrand, South Africa.

SM047, an IgM monoclonal antibody (Mab), was the product of hybridomacells derived by fusion of SP2 myeloma cells with splenic lymphocytes ofa mouse that had been immunized with a membrane preparation⁽⁷⁾ of anovarian serous cystadenocarcinoma, boosted once with cells from a cellline derived from that tumour and once with cells from the UWOV1 cellline which was derived from a similar tumour.⁽⁶⁾ The hybridomas wereproduced using essentially standard procedures with the exception thathuman umbilical cord serum replaced the fetal calf serum in the culturemedium. An ELISA assay was used to screen hybridoma-conditioned mediafor antibodies reactive with UWOV1 cells that had been cultured in96-well plates and fixed with 0.1% glutaraldehyde, as well as indirectimmunofluoresence with dried monolayers of the cells on multiwellTeflon-coated slides.⁽⁸⁾

b. Preparing Cell Lysates

Cells were washed twice with PBS and suspended at 4×10⁶/ml in lysisbuffer (PBS containing 0.5% Nonidet P-40, 1 mM phenylmethylsulphonylfluoride and 2 mM EDTA). Ater 20–30 min on ice the lysates wereconfigured at 12 000 g and the supernatant samples stored at −80° C.

c. Electrophoresis and Immunoblotting

Immunoprecipitates were boiled with reducing SDS sample buffer andelectrophoresed in polyacrylamide gels containing 0.1% SDS (SDS-PAGE),using the Laemmli buffer system.⁽⁹⁾ The resolved proteins weretransferred electrophoretically to PVDF membrane in a semidry apparatusin 0.025 M Tris, 0.192 M glycine in 20% methanol at 1 mA per cm² of gel.The membranes were dried overnight and processed for the identificationof Mab-reactive epitopes by blocking and incubating with Mab(hybridoma-conditioned medium 1:10). Bound antibody was detected byexposure to X-ray film after sequential treatment with biotinylatedrabbit anti-mouse immunoglobulin (0.55 μg/ml), streptavidin-peroxidase(0.16 μg/ml) and chemiluminescent substrate supplied byBoehringer-Mannheim. All dilutions were made in blocking solutionsupplied with the substrate and the blots were washed in Tris-bufferedsaline pH7.6 containing 0.05% Tween 20 between incubations. An IgManti-mycoplasma Mab (J138), raised in the laboratory, was used as acontrol antibody. Immunoprecipitates were analysed for sialylatedcarbohydrate constituents by SDS-PAGE, electroblotting to PVDF membranelabelling with biotin hydrazide after oxidation with sodiummetaperiodate at 4° C.⁽¹⁰⁾ according to the instructions of themanufacturers (AEC, Amersham, UK). The labelled carbohydrate wasdetected with streptavidin-peroxidase and chemiluminescent substrate asfor immunoblots.

d. Immunoprecipitation

Formalin-fixed Staphylococcus aureus bacteria (SA) at 10% w/v werewashed twice in PBS containing 0.5% Nonidet P-40 and 0.02% sodium azide(SA buffer), resuspended at 10% in SA buffer containing 1 mg/mlovalbumin (SA blocking solution) and incubated on ice for 30 min.One-tenth volume of rabbit anti-mouse immunoglobulin (at 15 mg/ml) wasadded and the SA left for a further 30 min on ice. The pellets were thenwashed twice with SA buffer, resuspended in 10× the original volume ofSM047 hybridoma conditioned medium and set on ice for a further 30 min.The SA pellet was again washed twice in SA buffer and resuspended at 10%in blocking solution. Cell lysates, sera or UWOV1-conditioned mediumwere immunoprecipitated with Mab-coated SA for 1 h on ice and washed 3×with SA buffer. Pellets were stored at −20° C. until SDS-PAGE.

EXAMPLE 2 The Preparation of Pathological Specimens

The surgical specimens used in the study for immunohistochemicalanalysis are shown in Table 1. Specimens had been fixed in 10%unbuffered formol saline or neutral buffered formol saline and routinelyprocessed in paraffin wax. Nine of the ovarian mucinous adenocarcinomaswere endocervical in type and the other 14 were enteric in type.

EXAMPLE 3 Immunohistochemical Staining and Analysis

Sections from paraffin wax-embedded blocks were cut ontoamino-propyltriethoxysilane-treated slides (Sigma, Poole, UK) and driedovernight at 37° C. Endogenous peroxidase activity was blocked in 3%alcoholic hydrogen peroxide for 10 min. Sections were pretreated in 0.1%trypsin in 0.1% calcium chloride at 37° C. and pH 7.8 or 8 for 10 or 20min (the immunostaining was performed at several different institutionsand was optimised in each of these laboratories. Sections were stainedusing the Mab SM047 at a dilution of 1:10. Localization was performedusing the peroxidase-streptavidin-biotin. Duet system (1:200; Dako,Glostrup, Denmark). Diaminobenzidine (Dako) was used as the chromagenand sections were counterstained using Harris' haematoxylin.Immunohistochemical staining was performed using appropriate positiveand negative controls. The positive control consisted of an ovarianserous adenocarcinoma with known diffuse strong membranousimmunoreactivity for SM047. For negative controls, the primary antibodywas omitted and replaced with mouse isotype immunoglobulin IgM (Dako) ata concentration equal to the primary antibody.

The intensity of staining was classified as negative, weak, moderate orstrong. Staining was also classified depending on the percentage ofcells exhibiting positivity as 0 (negative), +(0–25% of cells positive),++(26–50% of cells positive), +++(51–75% of cells positive) or ++++(>75%of cells positive). Positivity was further categorized as predominantlyinvolving the cell membrane or the cell cytoplasm.

Immunohistochemical Staining Results:

Table 1 shows the results of the immunohistochemical staining.Twenty-seven of 28 ovarian serous adenocarcinomas exhibited positivitywhich was usually strong and widespread and which involved the cellmembrane (FIG. 3). Positivity was most pronounced in well-differentiatedtumours and there was often no staining in poorly differentiated areas.The single ovarian serous adenocarcinoma which was completely negativewas extremely poorly differentiated. Moderate or strong positivemembranous staining was found in the five ovarian endometrioid and thesix clear cell adenocarcinomas. Sixteen of 23 primary ovarian mucinoustumours were completely negative, the others usually exhibiting focalweak positivity which was predominantly cytoplasmic. Three of the fourendocervical typers of ovarian mucinous adenocarcinomas were negative,the other case exhibiting positive membranous staining and 51–75% oftumour cells.

There was moderate or strong membranous staining of the 12 endometrialadenocarcinomas (10 endometrioid and two papillary serous) (FIG. 4) andof five of seven endocervical adenocarcinomas. There was positivestaining of some pancreatic (four of six), gastric (four of nine) andpulmonary adenocarcinomas (one of four).

Most other adenocarcinomas were completely negative, with the exceptionof six colonic adenocarcinomas, where staining was usually weak andcytoplasmic (FIG. 5). There was positive staining of four of 10mesotheliomas in addition to strong membranous staining of normalovarian surface mesothelium. There was no staining of negative controlmaterial.

TABLE 1 Specimens included in study together with immunohistochemicalstaining results Staining intensity Percentage positivity Tumour NegWeak Mod Strong 0 + ++ +++ ++++ Membranous Cytoplasmic Serousadenocarcinoma ovary 1 1 4 22 1 5 5 5 12 27 0 (η =28) Mucinousadenocarcinoma ovary 16 4 3 0 16 6 0 1 0 2 5 (η =23) Endometrioidadenocarcinoma ovary 0 0 1 4 0 2 1 1 1 5 0 (η = 5) Clear celladenocarcinoma ovary 0 0 1 5 0 2 1 1 2 6 0 (η + 6) Endometrioidendometrial adenocarcinoma 0 0 2 8 0 0 6 2 2 10 0 (η = 10) Papillaryserous carcinoma endometrium 0 0 0 2 0 0 0 0 2 2 0 (η = 2) Endocervicaladenocarcinoma 2 0 2 3 2 2 1 0 2 5 0 (η = 7) Colorectal adenocarcinoma15 4 0 2 15 1 1 2 2 2 4 (η = 21) Breast carcinoma 4 0 0 0 4 0 0 0 0 0 0(η = 4) Gastric adenocarcinoma 5 1 2 1 5 4 0 0 0 3 1 (η = 9) Prostaticadenocarcinoma 4 0 0 0 4 0 0 0 0 0 0 (η = 4) Pancreatic adenocarcinoma 20 0 4 2 3 1 0 0 4 0 (η = 6) Gallbladder adenocarcinoma 2 0 0 0 2 0 0 0 00 0 (η = 2) Lung adenocarcinoma 3 0 1 0 3 0 1 0 0 1 0 (η = 4)Mesothelioma 6 0 2 2 6 1 1 1 1 2 2 (η = 10)

EXAMPLE 4 Serological Test for the Detection of Sialylated SM047 Antigen

SM047-reactive molecules can be immunoprecipitated from the medium ofcultured ovarian carcinoma cells and from the serum of patients withcarcinoma of the ovary. Molecules bearing the SM047 epitope are alsopresent in the serum of healthy subjects but these differ from thetumour associated ones in that they are electrophoreticallyheterogeneous whereas those shed by ovarian tumours are homogeneous andsialylated. The diagnostic test utilizes this difference and detectsonly the sialylated molecules.

Wells of an ELISA plate are coated with SM047 antibody to capture thetumour-associated as well as normal SM047 antigen from patients' sera.The immobilized tumour antigen but not the normal antigen is thendetected by incubating with biotin-labelled lectin, which binds to thesialic acid residues on the tumour antigen, followed bystreptavidin-labelled horseradish peroxidase which binds to the biotin.The optical density of the coloured product produced by the peroxidasesubstrate is read with an ELISA reader.

The following reagents are required:

-   Purified monoclonal antibody SM047.-   Washing solution: phosphate-buffered saline (PBS) containing 0.05%    Tween 20.-   Blocking solution: 5% w/v skim milk powder in PBS.-   Sera from patients and normal volunteers, diluted 1:4 or more in    blocking solution.-   Detecting reagent: Maackia amurensis lectin labelled with biotin.-   Commercially available peroxidase-labelled streptavidin.-   Commercially available tetramethylbenzidine (TMB) peroxidase    substrate solution.-   Stopping solution: 1M phosphoric acid.

The following is, by way of example, one method of purification. TheSM047 hybridoma is grown in serum-free and protein-free medium (e.g.Sigma Hybrimax) and the spent medium collected. This can be concentratedby Amicon filtration to shorten the time required for loading thecolumn. A column of Hydroxyapatite (Bio-Gel HTP, BIO-RAD #130-0420, orequivalent) is prepared by resuspending the fully hydrated material in10 mM sodium phosphate pH 6.8 and a 5–10 ml column is prepared forprocessing about 500 ml conditioned medium. The column is washed with 10column volumes of 10 mM sodium phosphate pH 6.8. The antibody solutionis passed through the column and washed with 20 column volumes of 10 mMNa phosphate pH 6.8.

The antibody is eluted by raising the phosphate concentration in stepsof 50 mM, 100 mM, 200 mM and 300 mM (or by applying a linear gradient of120–300 mM Na phosphate pH 6.8). 1 ml fractions are collected and testedfor protein. IgM comes off in a sharp peak and can be stored in thephosphate buffer or further concentrated by ammonium sulphateprecipitation. The purity is checked by electrophoretic analysis(SDS-PAGE).

The column can be regenerated by washing in 1M NaCl in 10 mM Naphosphate pH6.8. The next step is to label the Maakia amurensis lectin(Sigma 18025 or equivalent) with biotin. Lectin is dissolved in 0.15MNaCl+0.1M NaHCO₃ at 2 mg/mL.

“Long-arm” biotin (Biotinamidocaproate-N-hydroxysuccinimide ester (SigmaB2643 or equivalent)) is dissolved at 2.5 mg/mL in dimethylsulphoxide(DMSO) and one-tenth volume of biotin solution is added to the lectinsolution, and the reaction is allowed at room temp. for 2 hours. Theproduct is dialysed against 0.2M NaCl+0.1M Tris pH7.4 and 0.1% sodiumazide added.

The tests are conducted in duplicate in Nunc “Maxisorp” ELISA plates.All reagents are used at 100 μL per well except the blocking solutionwhich is used at 250 μL per well. The plate is coated with purifiedSM047 antibody at 10 μg/mL in PBS and left overnight at 4 C. It is thenwashed three times. It is blocked for 1 hour at room temperature and theblocking solution then removed. The plate is incubated for 1 hour atroom temperature with diluted patients' or control sera and then washedthree times. It is then incubated for 1 hour at room temperature withbiotin-labelled lectin, diluted 1:1000 in PBS and again washed threetimes. Finally it is incubated for ½ hour at room temperature withstreptavidin-peroxidase diluted 1:1000 in PBS and then washed fivetimes. The TMB substrate is added and after 10 minutes at roomtemperature the phosphoric acid solution is added.

The results are shown in Table 2. The optical density is read at 450 nmwith an ELISA reader. In Table 2, the optical density readings for thedetected sialyated SM047 antigen are expressed as a percentage of a meanoptical density obtained for 5 normal control sera. The results forCA125 are given for comparison. As shown in Table 2, ovarian carcinomahas been proven in the patients A1 to A11.

TABLE 2 The serological test results SMO47 CA125 DIAGNOSIS A1 288% 347.3recurrent ca ovary (died) A2 280% 1069.4 recurrent serous cystadenoca ofovary A3 271% 350.3 residual serous cystadenoca of ovary A4 214% serousca ovary (died) A5 210% 106.6 recurrent serous cystadenoca of ovary A6180% 382.1 recurrent mixed epithelial cystadenoca of ovary A7 162% 59mucinous ca ovary, 10 days post-op A8 135% 202.3 residual serous caovary, post-op A9 103% ca uterus + ca ovary (endometrioid), 7d post-opA10  90% 257 mucinous cystadenoca of ovary, “intestinal variant” A11 56% 36.2 undifferentiated ca ovary

Immunoprecipitation Results:

In immunoprecipitation and immunoblots of lysates of cultured ovariancarcinoma cells SM047 bound molecules with an M_(r)>200 kDa (FIG. 1,lane 1). SM047 immunoprecipitates antigenic molecules with heterogeneouselectrophoretic mobilities from the sera of normal subjects and frompatients with ovarian carcinoma. Immunoprecipitates of SM047-reactivemolecules from ovarian carcinoma cell lysate and from normal andpatients' sera were analysed by SDS-PAGE in 4.5–15% gradient gels, ofwhich FIG. 1 is an example. After electrophoresis the precipitates weretransferred electrophoretically to a PVDF membrane and reacted withSM047 (FIG. 1, lanes 1–3) or J138 control antibody (lanes 4–6) followedby a peroxidase detection system with chemiluminescent substrate. Thecell lysate (lane 1) showed a distinct band which just entered theseparating gel, whereas in the normal serum (lane 3) antigers expressingthe SM047 epitope were heterogeneous and of lower M_(r), giving theappearance of a ‘smear’ in the Western blot. The serum from the patient(lane 2) contained both the high M_(r) and the lower M_(r) heterogeneousspecies, the latter being slightly reduced in quantity in the serum ofthe patient.

With this technique similar high M_(r) bands could be identified inseven of 15 sera from patients with ovarian malignancy. A homogeneous,high M_(r) band similar to that precipitated from the UWOV1 cell lysatewas also immunoprecipitated from the conditioned medium of these ovariancarcinoma cells (not shown), which confirms that the antigen is shedfrom the cells into the surrounding medium. The tumour-associatedantigen present in the serum of patients with carcinoma of the ovarycould thus be explained by shedding from the tumour into thecirculation.

The ovarian carcinoma-associated high molecular weight form of the SM047antigen is sialylated; the SM047 antigens in normal sera are not. SM047immunoprecipitates of pooled normal serum, of serum from a patient withovarian cancer and UWOV1 cell lysate were electrophoresed in a 7%acrylamide gel and transferred electrophoretically to PVDF membrane. Thetransferred antigens were then oxidized with metaperiodate at 4° C. andlabelled with biotin hydrazide to detect sialylated glycoprotein.⁽¹⁰⁾The biotin label was detected with streptavidin-peroxidase as in theimmunoblots. The result (FIG. 2) shows dearly that theimmunoprecipitated antigens from pooled normal serum were not sialylated(lane 1), whereas those from a patient's serum (lane 2) and carcinomacell lysate (lane 3) were.

The SM047 antigen has characteristics of a cell-associated proteoglycan.To analyse immunoprecipitates of metabolically labelled SM047 antigen,UWOV1 were incubated, either for 2 h or overnight, with ³⁵S-methionine,³H-leucine or ³⁵S-sulphate, in medium deficient of these precursors,using standard experimental protocols.⁽¹¹⁾ No detectable labelledantigen could be precipitated from the cell lysates labelled with³⁵S-methionine or ³H-leucine. On the other hand, lysates of UWOV1 cellsthat had been incubated overnight with ³⁵S-sulphate revealed ahomogeneous band of radioactive antigen similar to the sialylated tumourantigen when immunoprecipitated with SM047 and analysed by SDS-PAGE andfluorography (not shown).

The SM047 tumour antigen therefore has characteristics of acell-associated proteoglycan but it is not known if the normalheterogeneous form of the antigen is sulphated.

The above results show that there was strong positive membranousstaining of most non-mucinous ovarian adenocarcinomas but no staining ofthe majority of mucinous tumours with SM047. Moreover, mucinous tumours,when positive, usually exhibited weak cytoplasmic staining. Thisimmunohistochemical profile is similar to that of CA125 which is widelyused In pathological practice when attempting to confirm an ovarianprimary for an adenocarcinoma.

Such results suggest that SM047 staining is of value in the distinctionbetween a primary, ovarian endometrioid adenocarcinoma, which usuallyexhibits strong membranous positivity, and a colonic adenocarcinomawhich is usually either negative or exhibits weak cytoplasmicimmunoreactivity.

Adenocarcinomas of the stomach, pancreas and biliary tree whenmetastatic to the ovary may also closely resemble a primary ovarianadenocarcinoma,⁽¹²⁾ especially of mucinous type. With theseadenocarcinomas, the CK profile is similar to that of a primary ovarianadenocarcinoma. Most pancreatic adenocarcinomas studied exhibited strongmembranous staining with SM047 and the antibody is of value indistinguishing a primary ovarian mucinous tumour (usually SM047−) from ametastatic pancreatic adenocarcinoma (usually SM047+).

Diffuse involvement of the peritoneal cavity by ovarian serousadenocarcinoma or primary peritoneal serous adenocarcinoma may closelymimic mesothelioma clinically and histologically. Immunohistochemistrymay facilitate the distinction but there is considerable overlap. SM047is of some value, when used as part of a panel, since strong membranouspositivity is found in most serous adenocarcinomas whereas mostmesotheliomas are negative or exhibit cytoplasmic staining. However,occasional mesotheliomas exhibit strong membranous Immunoreactivity andthere is strong membranous staining of normal mesothelial cells.

CA125 is widely used as a serum marker of ovarian adenocarcinoma.⁽¹³⁾However, a raised serum CA125 is not specific for ovarianadenocarcinoma, this being found in a variety of conditions, especiallywhen there is widespread peritoneal disease. This can be attributed tothe production of CA125 by mesothelial cells.⁽¹⁴⁾ The SM047 antigenaccording to the invention was detected in sera of patients with ovarianmalignancy but also in normal human serum. However, there was aconsistent biochemical difference between normal and tumour-associatedSM047-reactive antigens, the former being non-sialylated and the lattersialylated.

In the present invention, it shows that most non-mucinous ovarianadenocarcinomas as well as adenocarcinomas primary elsewhere in thefemale genital tract are SM047+. Accordingly, the monoclonal antibodySM047 is useful when used as part of a larger panel, in determining thesite of origin of an adenocarcinoma.

REFERENCES

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1. A monoclonal antibody designated SM047, which is produced byhybridoma cells deposited with the ECACC, bearing Accession number01082905, and which is reactive against ovarian carcinoma.
 2. Ahybridoma cell producing a monoclonal antibody designated SM047, saidhybridoma cell deposited with the ECACC, bearing Access number 01082905,and said antibody reactive against ovarian carcinoma.
 3. A process forproducing a monoclonal antibody comprising culturing hybridoma cellsdeposited with the ECACC bearing Accession number 01082905 which producea monoclonal antibody designated SM047 and purifying the monoclonalantibody from the culture thus obtained.
 4. A diagnostic aid comprising:a) a monoclonal antibody designated SM047, which is produced byhybridoma cells deposited with the ECACC bearing the Accession number01082905 and b) a detectable label, whereby the binding of the antibodyin step (a) can be detected.
 5. A diagnostic aid as claimed in claim 4,wherein the label is selected from the group consisting of enzymes,radiolabels, chromophores and fluorescers.
 6. A diagnostic aid asclaimed in claim 4 for use in the detection of carcinoma.
 7. Adiagnostic aid as claimed in claim 6, wherein the carcinoma is anovarian carcinoma or female genital tract carcinoma.
 8. A method fordetecting carcinoma which comprises contacting a monoclonal antibodydesignated SM047, which is produced by the hybridoma cells depositedwith the ECACC, bearing Accession number 01082905, and which is reactiveagainst ovarian carcinoma with a human tissue or serum sample, anddetecting the interaction of said antibody with anyantigenically-corresponding carcinoma cells or antigenic determinants insaid sample.
 9. A method as claimed in claim 8, wherein the carcinoma isan ovarian carcinoma or female genital tract carcinoma.
 10. The methodas claimed in claim 8, wherein the interaction is detected byimmunohistological staining.
 11. A monoclonal antibody as claimed inclaim 1 for use in the manufacture of a diagnostic aid for detectingcarcinoma.
 12. A monoclonal antibody designated SM047, which is producedby hybridoma cells deposited with the ECACC, bearing Accession number01082905, and which is reactive against ovarian carcinoma supplied withinstructions for the use thereof in detecting carcinomas.
 13. Themonoclonal antibody as claimed in claim 12 wherein the instructions arein printed or written form.
 14. The monoclonal antibody as claimed inclaim 13 supplied in a package or container having said instructionsprovided therein or thereon.